The overall objective of this project is to investigate the molecular and structural mechanisms by which the mitotic apparatus regulates microtubule assembly and moves chromosomes. Our major approach is the development and analysis of in vitro mitotic model systems. We have recently developed a procedure for isolating and storing membrane-free mitotic spindles from marine eggs in a low ionic strength, calcium-chelating, detergent buffer. These isolated spindles are rapidly depolymerized by 1 uM calcium ions. Since microtubules polymerized in vitro from mammalian brain microtubule protein are not sensitive to such low concentrations of calcium, there may be a calcium-binding protein, perhaps calmodulin, associated with the spindle microtubules or a modification of the tubulin composition or structure that confers calcium lability of the spindle microtubules. We are using the isolated spindles as a model system to study the flux of tubulin through mitotic spindles and are looking specifically at how microtubule assembly is modified by colchicine and how fluorescently-tagged tubulin is incorporated and dissociates from spindle microtubules. We are now developing methods for isolating a mitotic apparatus that includes intact, functional membrane components as well as the fibrous structures of the mitotic spindle. We will use these models to test if the drugs thought to induce changes in microtubule assembly by causing the release of membrane-bound calcium actually function in such a manner. We are also investigating the coupling between microtubule assembly-disassembly mechanisms and the balance of forces in the mitotic spindle in living cells using hydrostatic pressure as a microtubule depolymerization agent.